N-methyl-D-aspartate-selective glutamate receptors (NMDA-Rs) mediate a slow component of excitatory synaptic transmission in the CNS and are involved in synaptic plasticity, learning, and memory. Activation of NMDA-Rs can contribute to the initiation and maintenance of seizures. In addition, stimulation of NMDA-Rs by extracellular glutamate that accumulates during ischemia can lead to cytotoxic levels of Ca2+ and neuronal death. Given this potential danger of NMDA-R overactivation, it is not surprising that NMDA-R function is tightly regulated by a number of endogenous extracellular ions including protons. Extracellular protons inhibit NMDA-R function completely by binding to a single binding site (Hill slope about 1) with a pKa of 7.0 (125 nM H+) or 7.4 (50 nM H+) for recombinant NRI/NR2A and NRI/NR2B receptors. Despite the potential importance of NMDA-R function, an understanding of the basic mechanisms by which NMDA-R channels open is lacking. No conceptual model exists for NMDA receptor function that explains both single channel and macroscopic receptor properties. The experiments outlined for the next period exploit our recent success obtaining excised outside-out patches that contain only one active NMDA-R channel. This single channel approach will be combined with macroscopic current recording and quantitative modeling to explore the mechanism of NMDA-R activation (spec. aims 1-3). Detailed functional information about NMDA-R gating will be required to maximize interpretation of structural information. Understanding NMDA-R gating is also a pre-requisite to understanding both the proton sensitivity of gating and the function of the therapeutically interesting compounds that regulate proton inhibition (aims 4-5). Five questions will be addressed. I. Is NMDA receptor function controlled by two independent gates? 2. Can single channel kinetics and macroscopic current response time course be reconciled by multi-gate models? 3. Do the glycine and glutamate binding subunits contribute kinetically distinct gates to the NMDA receptor pore? 4. Do protons and phenylethanolamines reduce the probability that an agonist-bound receptor will open? 5. Is the structural basis for H+ sensitivity of NMDA and G1uR6 receptors contained in the transmembrane linker regions? Together, these experiments will help define a unifying theory for NMDA receptor function that accounts for single channel and macroscopic behavior. In addition, evaluation of the hypothesis that protonation of a few key residues inhibits channel opening without changing other features of receptor function will increase our understanding of the structural nature of how glutamate receptors open and close in response to full and partial agonists. These experiments also probe the link between gating of NMDA receptors and inward rectifier K+ channels. Finally, we will determine at the single channel level the mechanisms of action of a non-competitive and therapeutically interesting class of antagonists - phenylethanolamines, such as ifenprodil.